Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

The effective design and control of a capillary electrophoresis (CE) microchip requires a thorough understanding of the electrokinetic transport phenomena associated with its microfluidic injection system. The present study utilizes a numerical simulation approach to investigate these electrokinetic transport processes and to study the control parameters of the injection process. Injection systems with a variety of different configurations are designed and tested, including the cross-form, T-form, double-T-form, variable-volume focused flow cross-form, and variable-volume triple-T-form configuration. Each injection system cycles through a predetermined series of steps in which the magnitudes and distributions of the applied electric field are precisely manipulated in order to effectuate a virtual valve. This study investigates the sample leakage effect associated with each of the injection configurations and applies the double-L, pullback, and focusing injection techniques to minimize the sample leakage effect. The injection methods presented in this paper have the exciting potential for use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis

In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%.     

Multiple injection techniques for microfluidic sample handling

This paper presents an experimental and numerical investigation into electrokinetic focusing flow injection for bioanalytical applications on 1 x N (i.e., 1 sample inlet port and N outlet ports) and M x N (i.e., M sample inlet ports and N outlet ports) microfluidic chips. A novel device is presented which integrates two important microfluidic phenomena, namely electrokinetic focusing and valveless flow switching within multiported microchannels. The study proposes a voltage control model which achieves electrokinetic focusing in a prefocusing sample injection system and which allows the volume of the sample to be controlled. Using the developed methods, the study shows how the sample may be prefocused electrokinetically into a narrow stream prior to being injected continuously into specified outlet ports. The microfluidic chips presented within this paper possess an exciting potential for use in a variety of techniques, including high-throughput chemical analysis, cell fusion, fraction collection, fast sample mixing, and many other applications within the micrototalanalysis systems field.     

A low-leakage sample plug injection scheme for crossform microfluidic capillary electrophoresis devices incorporating a restricted cross-channel intersection

This study develops a crossform CE microfluidic device in which a single-circular barrier or a double-circular barrier is introduced at the cross-channel intersection. Utilizing a conventional crossform injection scheme, it is shown that these barriers reduce sample leakage and deliver a compact sample band into the separation channel, thereby ensuring an enhanced detection performance. A series of numerical and experimental investigations are performed to investigate the effects of the barrier type and the barrier ratio on the flow streamlines within the microchannel and to clarify their respective effects on the sample leakage ratio and sample plug variance during the injection process. The results indicate that a single-circular barrier injector with a barrier ratio greater than 20% and a double-circular barrier injector with a barrier ratio greater than 40% minimize the sample leakage ratio and produce a compact sample plug. As a result, both injectors have an excellent potential for use in high-quality, high-throughput chemical analysis procedures and in many other applications throughout the micro-total analysis systems field.     

Experimental and numerical investigation into leakage effect in injectors of microfluidic devices

This paper performs an experimental and numerical investigation into low-leakage injectors designed for electrophoresis microchips. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered in developing a mathematical model of the electrophoresis process. Low-leakage injectors designed with injection channels orientated at various included angles are designed and tested. The numerical and experimental results indicate that the injector with a 30 degrees included angle successfully minimizes sample leakage and has an exciting potential for use in high-quality, high-throughput chemical analysis procedures and in many other applications in the field of micro-total analysis systems.     

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 μm, respectively. The separation was detected by CCD (charge-coupled device) (effective LENGTH=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2–5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.     

Optimal configuration of capillary electrophoresis microchip with expansion chamber in separation channel

This study develops a novel capillary electrophoresis (CE) microfluidic device featuring a conventional cross-form injection system and an expansion chamber located at the inlet of the separation channel. The combined injection system/expansion chamber arrangement is designed to deliver a high-quality sample band into the separation channel such that the detection performance of the device is enhanced. Numerical simulations are performed to investigate the electrokinetic transport processes in the microfluidic device and to establish the optimal configuration of the expansion chamber. The results indicate that an expansion chamber with an expansion ratio of 2.5 and an expansion length of 500 microm delivers a sample plug with the correct shape and orientation. With this particular configuration, the peak intensities of the sample are sharp and clearly distinguishable in the detection region of the separation channel. Therefore, this configuration is well suited for capillary electrophoresis applications which require a highly sensitive resolution of the sample plug. The novel CE microfluidic device developed in this study has an exciting potential for use in high-performance, high-throughput chemical analysis applications and in many other applications throughout the field of micro-total-analysis-systems.     

Sample introduction techniques for microchip electrophoresis: A review

The number of applications of microfluidic analysis systems continues to increase, along with the variety of substrate materials and complexity of the devices themselves. One of the most common features of these devices that has remained relatively unchanged, however, is the introduction of a sample mixture into a separation channel so that individual components can be separated by electrophoresis. Whether a relatively simple mixture of amino acids or a more complex sample of DNA fragments extracted and amplified on-chip, the ability to reliably and reproducibly inject a representative sample is arguably the most significant requirement for an electrophoretic micro total analysis system (μTAS). This review will focus on the different methods reported for sample introduction in microchip electrophoresis, highlighting both pressure-driven and electrokinetic techniques, with an emphasis on the methods employed in μTAS applications.     

Design of an interface to allow microfluidic electrophoresis chips to drink from the fire hose of the external environment

An interface design is presented that facilitates automated sample introduction into an electrokinetic microchip, without perturbing the liquids within the microfluidic device. The design utilizes an interface flow channel with a volume flow resistance that is 0.54-4.1 x 10(6) times lower than the volume flow resistance of the electrokinetic fluid manifold used for mixing, reaction, separation, and analysis. A channel, 300 microm deep, 1 mm wide and 15-20 mm long, was etched in glass substrates to create the sample introduction channel (SIC) for a manifold of electrokinetic flow channels in the range of 10-13 microm depth and 36-275 microm width. Volume flow rates of up to 1 mL/min were pumped through the SIC without perturbing the solutions within the electrokinetic channel manifold. Calculations support this observation, suggesting a leakage flow to electroosmotic flow ratio of 0.1:1% in the electrokinetic channels, arising from 66-700 microL/min pressure-driven flow rates in the SIC. Peak heights for capillary electrophoresis separations in the electrokinetic flow manifold showed no dependence on whether the SIC pump was on or off. On-chip mixing, reaction and separation of anti-ovalbumin and ovalbumin could be performed with good quantitative results, independent of the SIC pump operation. Reproducibility of injection performance, estimated from peak height variations, ranged from 1.5-4%, depending upon the device design and the sample composition.     

Electrokinetic injection in capillary electrophoresis and its application to the analysis of inorganic compounds

In capillary electrophoresis, electrokinetic injection is a highly controversial sampling technique. It is a simple mode of sample introduction which is suitable for on-line preconcentration of the analytes, but its precision and accuracy are more strongly affected by experimental conditions compared to hydrodynamic injection. In the first part of this paper the features of electrokinetic and hydrodynamic injections are compared, followed by a detailed discussion on the different biases of electrokinetic injection and on how to reduce them. Finally, applications of the electrokinetic injection are reviewed with special emphasis on the analysis of inorganic compounds.     

Optimization of sample transfer in two-dimensional microfluidic separation systems

Multidimensional microfluidic separation systems combining a first dimension microchannel with an array of parallel second dimension microchannels can suffer from non-uniform sample transfer between the dimensions, sample leakage, and injection plug tailing within the second dimension array. These factors can significantly reduce overall two-dimensional separation performance. In this paper, numerical and analytical models reveal an optimized chip design which combines multidimensional backbiasing and an angled channel geometry to ensure leakage-free and uniform interdimensional sample transfer, while also minimizing injected sample plug lengths. The optimized design is validated experimentally using a multidimensional chip containing five second dimension channels.     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Microfluidic chip electrophoresis for biochemical analysis

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics‐based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.     

Improvements on the electrokinetic injection technique for microfluidic chips

This paper presents a T-form electrokinetic injection system for the discrete time-based loading and dispensing of samples of variable-volume in a microfluidic chip. A novel push-pull effect is produced during the loading and dispensing processes by the application of an appropriate control voltage distribution. The experimental and numerical results show that this push-pull loading technique produces compact sample plugs and hence improves the detection resolution of the microfluidic device. The injection system is integrated with a microflow switch, and a suitable voltage control scheme is proposed to guide the sample to the desired outlet port such that the microfluidic device can function as a microdispenser. The time-based variable-volume T-form injection method presented in this study is performed using a compact geometry and a simple control scheme and can be readily integrated with other microfluidic devices to form a microfluidic system capable of continuous monitoring and analysis of bioreactions in the life science and biochemistry fields.     

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

Bias-free pneumatic sample injection in microchip electrophoresis

We have developed a new microfluidic chip capable of accurate metering, pneumatic sample injection, and subsequent electrophoretic separation. The pneumatic injection scheme, enabling us to introduce a solution without sampling bias unlike electrokinetic injection, is based upon the hydrophobicity and wettability of channel surfaces. An accurately metered solution of 10 nL could be injected by pneumatic pressure into a hydrophilic separation channel through Y-shaped hydrophobic valves, which consist of polydimethylsiloxane (PDMS) and fluorocarbon (FC) film layers. We demonstrated the successful pneumatic injection of a red ink solution into the separation channel as a proof of the concept. A mixture of fluorescein and dichlorofluorescein (DCF) could be baseline-separated using a single power source in microchip electrophoresis.     

Characterization of electrokinetic gating valve in microfluidic channels

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.     

Numerical analysis of an electrokinetic double-focusing injection technique for microchip CE

The injection techniques in electrophoresis microchips play an important role in the sample-handling process, whose characteristics determine the separation performance achieved, and the shape of a sample plug delivered into the separation channel has a great impact on the high-quality separation performance as well. This paper describes a numerical investigation of different electrokinetic injection techniques to deliver a sample plug within electrophoresis microchips. A novel double-focusing injection system is designed and fabricated, which involves four accessory arm channels in which symmetrical focusing potentials are loaded to form a unique parallel electric field distribution in the intersection of injection channel and separation channel. The parallel electric field effectuates virtual walls to confine the spreading of a sample plug at the intersection and prevents sample leakage into separation channel during the dispensing step. The key features of this technique over other injection techniques are the abilities to generate regular and nondistorted shape of sample plugs and deliver the variable-volume sample plugs by electrokinetic focusing. The detection peak in the proposed injection system is uniform regardless of the position of the detection probe in the separation channel, and the peak resolution is greatly enhanced. Finally, the double-focusing injection technique shows the flexibility in detection position and ensures improved signal sensitivity with good peak resolution due to the delivered high-quality sample plug.